Lysosome-associated membrane protein-1 (LAMP-1) is a type I membrane glycoprotein with a COOH-terminal cytoplasmic tail (CT) containing a lysosome-targeting signal of GYQTI³⁸²-COOH. This sequence is categorized as a tyrosine-based motif of GYXXΦ where Φ is a bulky hydrophobic amino acid residue. Lysosomal localization of LAMP-1 varies by changing the COOH-terminal amino acid residues. Adaptor protein (AP) complexes are reported to recognize the tyrosine-based signal peptide for efficient lysosomal transport of LAMP-1. In order to better understand the role of APs in lysosomal transport of LAMP-1, we have studied interactions of wild-type (WT) and mutated CTs of LAMP-1 with medium (μ) subunits of the four APs by a yeast two-hybrid (Y2H) system and subsequent computer-based molecular modeling. Among the μ subunits of AP-1, AP-2, AP-3 and AP-4, called μ1, μ2, μ3A, and μ4, respectively, the WT-CT significantly interacted with μ3Α in the Y2H system. The degree of interaction of the WT and mutated CTs with μ3A from the Y2H analyses correlated with that of their dissociation constants determined by computer-based molecular modeling, and also with that of the late endosomal and lysosomal amount of WT and the similar mutants of LAMP-1. These results reinforce the notion that AP-3 makes a major contribution to the vesicular transport of LAMP-1 to late endosomes and lysosomes via a direct intracellular route.